The corneal tissue consists of five layers, a corneal epithelium layer on the outermost surface, Bowman's membrane, a corneal stromal layer, Descemet's membrane, and a corneal endothelium layer. The innermost corneal endothelium layer serves as a liner on the corneal tissue, and Descemet's membrane corresponds to the basement membrane of the corneal endothelial layer which is composed of adhesive proteins. The function of corneal endothelial cells is to pump water out of the corneal stroma into the anterior chamber counteracting the swelling pressure of the stroma, presumably due to the pumping action as assisted by Na—K ATPase. Human corneal endothelial cells will not usually undergo cell division in the living body, and denatured or exfoliated endothelial cells are compensated for by the thickening or migration of the remaining cells. If the remaining corneal endothelial cells are too few, the pumping function of the corneal endothelial tissue is no longer adequate and the corneal tissue swells to develop diseases such as corneal endothelial disorder and bullous keratopathy. Therapies of these diseases include the use of therapeutic soft contact lenses to deal with eye pain and the use of ophthalmic ointments and eye drops of hypertonic saline in order to remove water from the swollen corneal tissue; however, these methods are just symptomatic and radical therapies that affect the basic underlying causes of the symptoms have been desired.
With marked advances in medical technology, it has recently become popular to perform organ transplants, i.e., replacing a difficult-to-treat organ with another person's organ. This is also true with diseases such as the above-mentioned corneal endothelial disorder and bullous keratopathy and attempts are being made to treat them radically by transplanting all layers of the cornea. However, the number of donors in Japan is still considerably smaller than that of patients and while there are annually about 20,000 patients who need keratoplasty, only a tenth of them (ca. 2,000 in number) can actually be treated by that procedure. Although keratoplasty is a virtually established procedure, it suffers the problem of shortage in donors, giving rise to the need for the development of a next-generation medical procedure.
As a means of solving this problem, the technology of regenerative medicine which involves obtaining a required tissue by artificial in vitro cultivation has recently seen a rapid advance. Conventionally, such cell culture has been performed either on the surface of glass or on the surface of synthetic polymers that were subjected to a variety of treatments. For example, a variety of polystyrene vessels that were subjected to surface treatments such as γ-ray irradiation and silicone coating have become popular for use in cell culture. However, the above-mentioned corneal endothelial cells are known to be cells that are difficult to grow in high density on the surfaces of such vessels and a better method of culture has been desired.
Cells that have been cultivated to grow on vessels for cell culture are detached and recovered from the surfaces of the vessels by treatment with proteolytic enzymes such as trypsin or chemical reagents. However, it has been pointed out that the recovery of grown cells by treatment with chemical reagents involves some disadvantages such as an increased chance of contamination by impurities and the grown cells becoming denatured or damaged by the chemical treatment to have their inherent functions impaired. In order to overcome these disadvantages, several techniques have been proposed to date.
JP 2-23191 B describes a method for producing a transplantable membrane of keratin tissue which comprises the steps of cultivating human neonatal keratinized epidermic cells in a culture vessel under conditions that enable a membrane of keratin tissue to form on the surface of the vessel and detaching the membrane of keratin tissue using an enzyme. Specifically, with 3T3 cells used as a feeder layer, the epidermic cells are grown and stratified as a cell sheet which is recovered using the proteolytic enzyme dispase. However, the method described in JP 2-23191 B has had the following defects.
(1) Dispase is of microbial origin and the recovered cell sheet needs to be washed thoroughly.
(2) The conditions for dispase treatment differ from one batch of cell culture to another and great skill is required in the treatment.
(3) The cultured epidermic cells are pathologically activated by dispase treatment.
(4) The extracellular matrix is decomposed by dispase treatment.
(5) As the result, the diseased part to which the cell sheet has been grafted is prone to infection.
In addition to these defects of the prior art method, corneal endothelial cells that are contemplated in the present invention do not have as strong intercellular binding as dermal cells and have had the problem that cultivated cells cannot be detached and recovered as a single sheet even if the dispase is employed.
In Japanese Patent Application No. 2001-226141, anterior segment related cells are cultivated on a cell culture support comprising a substrate having its surface coated with a temperature responsive polymer having an upper or lower critical temperature of 0-80° C. at which it dissolves in water and, if necessary, the cultured cell layer is stratified by the usual method and the cultured cell sheet is detached by merely changing the temperature of the support. The detached cell sheet has adequate strength. However, considering the take and functions of the corneal endothelial cell sheet that is actually obtained, further improvements have been desired.